Experimental diabetic neuropathy is characterized by a variety of biochemical and functional alterations, including reduced peripheral nerve conduction velocity, elevated sorbitol levels, decreased myo- inositol content and diminished Na+,K+-ATPase activity. The pathogenesis of this disorder remains obscure and is likely caused by complex multifactorial processes. In recent years, our laboratory has shown that in sciatic nerve from diabetic rats both phosphatidylinositol-4,5- biphosphate (PIP2) monoesterified phosphate group turnover and myelin protein phosphorylation are increased, whereas agonist-stimulated phospho-inositide breakdown exhibits a blunted response. In addition, diabetic nerve contains a reduced amount of arachidonic acid (AA)- containing molecular species in phospholipids and 1,2-diacylglycerol. The overall goal of this project is to evaluate the hypothesis that these and other signal transduction-related events are critically involved in the onset of peripheral nerve dysfunction. Four specific aims are proposed to achieve this objective: (1) We will examine the extent, severity and reversibility of altered basal and agonist-stimulated phosphoinositide metabolism, protein phosphorylation and Na+ pump activity in nerves from rats treated with high doses of streptozotocin and maintained on minimal insulin therapy so as to induce extreme and variable hyperglycemia similar to poorly controlled human diabetes. The biochemical measurements will be correlated with evaluation of functional and morphological abnormalities. (2) We will study the mechanisms underlying possible alterations in diabetic nerve of several signal transduction-associated events, including: (a) GTPgammaS-stimulated PIP2 hydrolysis in solubilized myelin-enriched preparations; (b) levels, subcellular distribution and phorbol ester-mediated downregulation of total protein kinase C (PKC) activity and PKC isoforms; (c) the phorbol ester-stimulated tyrosine phosphorylation of a ca. 30 kDa nerve protein, provisionally identified as the major peripheral myelin protein, Po, but possibly another molecule. (3) We will determine whether the deficit in AA-containing molecular species is relieved when diabetic rats are fed a diet supplemented with gamma-linolenic acid, a precursor of AA, and concomitantly if prostaglandin levels and biosynthesis are affected and conduction velocity is corrected. We will also assess whether treating rats with a cyclooxygenase inhibitor will antagonize any beneficial effects obtained. (4) We will investigate signal transduction events in Schwann cells derived from adult rats to determine: (a) whether these alterations appear when Schwann cells from normal animals are maintained in elevated glucose concentrations; (b) which abnormalities persist in cells from diabetic rats cultured in physiological glucose levels; (c) if the changes can be reversed by insulin in the culture medium. These studies should furnish new information relevant to the etiology of experimental diabetic neuropathy and which may bear on the pathogenesis of the human disorder.